Sarcoplasmic Hypertrophy
A changing opinion
A study was recently published supporting the idea of sarcoplasmic hypertrophy [Haun et al., 2019]. This post is a product of the discussion on the Stronger by Science Podcast where I was hesitant, but agreed that sarcoplasmic hypertrophy can occur. In this review I’ll go through the data and help shed some light on the findings.
I went on the Revive Stronger podcast to discuss this idea if you want to listen after you read this.
What is hypertrophy?
The traditional thought is that hypertrophy occurs through increases in muscle fiber size with an increase in myofibrillar proteins (from resistance training). Therefore, we will see an increase in the muscle fiber cross sectional area (fCSA) due to an increase in predominantly myosin and actin.
“… muscle fibers may increase in diameter, as is found in singly innervated muscles, to increase the number of myofibrils in parallel...”-[Paul and Rosenthal 2002]
“…in response to elevated forces, more sarcomeres, the force-producing units of muscle, are built and added in parallel, increasing muscle cross sectional area.. -Wisdom et al. (2015)”
What is sarcoplasmic hypertrophy?
We previously defined it as “…an increase in the volume of the sarcolemma and/or sarcoplasm accompanied by an increase in the volume of mitochondria, sarcoplasmic reticulum, t-tubules, and/or sarcoplasmic enzyme or substrate content.”- [Haun et al., 2019]
Why is sarcoplasmic hypertrophy important?
Sarcoplasmic hypertrophy is making a muscle larger without adding myofibrillar proteins like actin and myosin. This could mean training in ways where metabolic stress cause increases in muscle size. We could, presumably, use higher repetition training to cause adaptations to occur like increases in other the sarcoplasm to buffer the metabolic stress.
Disclaimer: I’ve previously published with this lab.
First, let’s orient ourselves with a muscle fiber.
In a muscle cell we have a number of major components: nuclei, mitochondria, glycogen, myofibrils, and sarcoplasm. Each muscle fiber has myofibrils, which contain myosin and actin — the proteins we care about for producing force.
Now, let’s transition to the publication on sarcoplasmic hypertrophy…
Study: Haun et al., (2019)
Purpose: To determine if training affected actin and myosin protein concentrations, sarcoplasmic protein concentrations, glycogen concentrations, and mitochondrial volume.
Hypothesis: Individuals experiencing notable fCSA increases would experience a decrease in myosin and actin concentrations, a decrease in citrate synthase activity, and either no change or an increase in sarcoplasmic protein and glycogen concentrations.
Study Summary: These data challenge current dogma suggesting fCSA increases during high-volume resistance training are primarily driven through increases contractile protein content. The authors interpret the data to suggest sarcoplasmic hypertrophy is largely responsible for short-term fCSA increases.
Let’s dive deeper.
All of the subjects were from a previous study, which is discussed briefly at the very end of this article. In the current study, there were 15 responders measured by change in mean fiber cross-sectional area (fCSA). The participants used in the study were a subset of responders who increased fCSA as indicated in the white box.
Here’s how the fCSA, fat mass (FM) and fat-free mass (FFM) compared with those subjects in a response heterogeneity plot. You can see that those who gained the most FFM tended to either lose FM or not change much. You can also see that those who had the largest fCSA change tended to have smaller change in FFM. We covered measurement issues in the aforementioned manuscript where it was concluded that— “..different assessment techniques seem to disagree with one another, we posit that this conundrum provides tremendous opportunity for future researchers to build upon current methods or generate newer and more valid methods to better assess skeletal muscle hypertrophy.” Importantly, the data below isn’t correct for total body water which is an important limitation.
Let’s go through a few key results from the study:
1. There were no changes in glycogen content.
Glycogen is distributed within the muscle fibers in the subsarcolemma (5–15 %) intermyofibrillar (~75 %) and intramyofibrillar (5–15 %). Practically, this means it is stored within and between the myofibers. When we resistance train we use muscle glycogen as a fuel source, but rarely more than ~30–40% (Cholewa et al., 2019). The lack of a change in glycogen isn’t surprising given these were well-trained subjects, but then again, there is very little data on glycogen changes with chronic RT. I actually couldn’t find any direct data. I will admit that I thought some of the sarcoplasmic changes could be due to increases in glycogen — apparently not.
2. There were no changes in myosin protein content.
Myosin is one of the main proteins involved in muscle contractions. In the original analysis there were no statistically significant changes in myosin and actin protein concentrations (p = 0.052 Fig 1G–1I). However, given that both p-values approached statistical significance, “forced” post hoc tests were performed to see where the differences would be. These forced post hoc tests indicated myosin and actin protein concentrations significantly decreased from PRE to W6 (p = 0.035 for each target). Another method they could have used, only looking at pre and post is a paired t-test, which would have been significant (p= 0.035). Alternatively, planned contrasts for Week 0 and Week 6 could have been done pre-analysis, especially after finding the odd fCSA data in the other study. Independent of what analysis we use; this data supports the idea that myosin is decreasing in these participants.
I’ve graphed the data without the three week timepoint. I love that they included the midpoint (week 3) in the original analysis. However, I wanted to look at the data a few different ways to visualize it because of the low sample size, especially since we know there is something odd going on at that week 3 timepoint. We also wouldn’t expect much hypertrophy in 3 weeks in trained participants. This data left me a little skeptical because there are a few people who seem to increase myosin protein content, so I regraphed the data a different way.
Here I used percent change from Week 0. We can now easily see that only three participants increased MHC. Therefore, I think we can comfortably say that myosin protein was decreased in most of the participants.
3. There were no changes in actin protein content.
The actin data was very similar to the myosin data, so I didn’t graph all of it, but the take-home is the same. There appears to be a decrease in actin.
The authors also found no changes in actin via phalloidin staining, but I’m not super confident in that method due to the flourescent images. I generally see a ton of background when using green with muscle histology.
3. There were increases in sarcoplasmic protein content.
This finding solidified that something was occuring in the sarcoplasm. In lab, we can use methods to separate the muscle into different fractions using buffers and centrifugation. Centrifugation separates proteins by weight, so that heavier proteins move towards the bottom. Afterward, we can identify the protein content within each fraction. This data indicate that protein content increased in the sarcoplasm. Therefore, proteins in the sarcoplasm were increasing in half the participants.
4. There were decreases in mitochondria.
There was a ~24% decrease in citrate synthase activity, which is a well accepted marker for mitochondria. Citrate synthase activity increases with endurance, high-intensity interval training, or resistance training (Porter et al., 2016) although few studies have been completed in subjects this highly trained. However, another study found a slightly lower level of citrate synthase activity in resistance trained athletes compared to controls (Salvadego et al., 2013) so the idea has some support in the literature. I also thought mitochondria would increase with this type of training—wrong again.
5. The proteomics data show an increase in sarcoplasmic proteins.
Proteomics is the large-scale study of proteins. It’s an easy way to look at a lot of proteins at once and is increasingly used to analyze complex protein populations in biological samples such as skeletal muscle.
Once you get data we can then use bioinformatics techniques to help interpret it.
Here, the authors used a pathway analysis (DAVID v6.8) to show increases glycolysis, acetylation, gluconeogenesis, and cytoplasmic proteins. They also used a KEGG pathway analysis which indicated that glycolysis/gluconeogenesis (8 up-regulated proteins) was significantly up-regulated from Week 0 to Week 6.
This is where things get really fun since I have a good bit of experience analyzing -omics type data. I pulled down their dataset and ran it through a few different analysis.
Reactome
First I ran an overrepresentation analysis: A statistical (hypergeometric distribution) test that determines whether certain Reactome pathways are over-represented (enriched) in the data. It answers the question ‘Does my list contain more proteins for pathway X than would be expected by chance?’ This test produces a probability score, which is corrected for false discovery rate using the Benjamani-Hochberg method. When we do a lot of comparisons we need to correct for the chance they could be false-positives or false-negatives. Here’s the results from timepoint 1 (Week 0) and timepoint 3 (Week 6). One of the major takeaways is how much metabolism pops out at week 6, which confirms what the authors found.
Another analysis essentially shows the same thing. Metabolism is highly upregulated post-training.
I found the exact same thing as the authors using different methods. That’s known as reproducibility and I think the authors publishing raw data sets should be highly applauded. Most scientists won’t do that.
In summary, we have:
- An increase in muscle fiber size
- No changes in glycogen
- A decrease in myosin and actin
- An increase in sarcoplasmic proteins
- A decrease in mitochondria
- An increase in metabolic and sarcoplasmic proteins
Therefore, we have a few things that fit our definition of sarcoplasmic hypertrophy but not everything.
Definition: Sarcoplasmic hypertrophy is an increase in the volume of the sarcolemma and/or sarcoplasm accompanied by an increase in the volume of mitochondria, sarcoplasmic reticulum, t-tubules, and/or sarcoplasmic enzyme or substrate content.
How does the data relate to the literature?
The authors do an excellent job of relating their data to the literature. Here are some quotes from the discussion with data from the cited papers and a quick summary.
Study [16] = Penman 1969
Quote: a study which examined muscle fiber ultrastructural alterations in humans in response to isotonic, isometric, and run training reported decreases in the number of myofibrils per muscle fiber area across all training conditions as assessed by transmission electron microscopy (TEM) [16].
Summary: This study was tiny so I’m hesitant to even mention it. Penman found an increase in glycogen, intracellular fats, greater mitochondria, decreased myosin density, and an increase in myosin fiber diameter with resistance training. I didn’t extract the data because of the extremely low sample size (n=2). However, I added an image of a muscle biopsy taken with an electron microscope so you can see some of the changes we’re trying to identify.
Study [17] = MacDougall et al., 1982
Quote: Another study utilizing TEM [17] similarly reported space occupied by myofibrils decreased and space occupied by sarcoplasmic area increased in previously untrained humans following six months of resistance training in lieu of significant increases in fCSA.
Summary: This study found a decrease in myofibrillar volume and mitochondrial density. Furthermore, bodybuilders/powerlifters (far right bars) had even lower myofibrillar volume and mitochondrial density compared to participants that were trained for 6 months, which further makes me think that sarcoplasmic hypertrophy can occur — especially since the sarcoplasm is extremely elevated in these participants compared to the other data.
Study [18]: Toth et al., 2012
Quote: A similar study utilizing TEM [18] reported that 18 weeks of resistance training resulted in a ~15% decrease in space occupied by myofibrils in both heart failure patients and healthy controls.
Summary: Resistance training results in a loss of myofibrillar area in healthy participants. I left out the data on heart failure patients because there are a lot of other potential issues with that could interfere with the interpretation.
Study not mentioned: Claassen et al., 1986
Quote: The mean linear distance between myosin filaments was similar before and after training when estimated from measurements of packing density or from direct measurements of the intermyosin filament distance. We also found no significant changes in the actin to myosin ratio withtraining.
Summary: This study wasn’t cited, but it also wasn’t cited in our hypertrophy review nor was I aware of it before looking into the literature. Interestingly, this supports the idea of myofibrillar packing. Can this translate to why people look “more dense” as they train longer? I’m not sure, but it might. Importantly, this data shows decreases in mitochondria and sarcoplasm post-training, which match some of the data shown above.
Literature Comparison
Compared to the literature we have one study that’s too small [Penman 1969], two that agree with some of the data [Toth et al., 2012; Claassen et al., 1986], and one that has very similar data in a similar population [MacDougall et al., 1982].
Can sarcoplasmic hypertrophy occur?
Probably. However, as the authors note in the manuscript, it may be a transient response to training and evidence is still limited. There needs to be more research to better understand the physiology.
Is sarcoplasmic hypertrophy something to target with training?
No. We just don’t have enough research to support specific training methods to target sarcoplasmic hypertrophy. It seems that higher volume training could help, but training in multiple repetition ranges is the safest bet for promoting muscle growth.
Where else can I learn about sarcoplasmic hypertrophy?
Dr. Nedergaard wrote about it in 2014.
Greg Nuckols covered sarcoplasmic hypertrophy in depth back in 2015 on Stronger by Science.
Dr. Galpin covered it in a recent podcast entitled Muscle Growth on the Body of Knowledge Podcast.
Keep reading for a brief summary of the original study….
The original study:
To give some context, the data above is from a secondary analysis of Haun et al., 2018 wherein three different groups of well-trained, college-aged males, performed 6 weeks of resistance training. The frequency was 3 days per week and each session involved 2 upper- and 2 lower-body exercises (10 repetitions/set). Volume increased from 10 sets/exercise (week 1) to 32 sets/exercise (week 6).
I think one of the most interesting findings from the original study was this:
There were differential hypertrophy responses in the biceps and vastus lateralis (muscles) as assessed via ultrasound. They found increases in biceps thickness at the 3-week timepoint. Yet, they found increases in VL from 3 weeks to 6 weeks. When we think about muscle hypertrophy — we think it’s linear. Maybe that’s not the case. Yet, a more simple explanation is muscle swelling or shifts in fluid due to the high training volume, which the authors discuss in the manuscript.
If we look at the biopsy data to see what occurred at the muscle fiber level, it appears that subjects lost muscle fiber size at the midpoint and had a rebound at the end of the study in the VL, which parallels the ultrasound data. This data could indicate a transient contraction or expansion of the fiber. We’ve rarely seen this in the literuature and I plan to dig into this idea in a future post. Basically, I think there might be some transient changes that we’ve missed because of the timepoints chosen in most resistance training studies.
